Use of second steroid to accelerate 1-dehydrogenation of substrate steroid



Uted States Patent 2302, 111 Patented Sept. l, 1953 USE OF SECOND STEROID TO ACGELERATE I-DE- HYDROGENATION F SUBSTRATE STEROID No Drawing. Application June 28, 1957 Serial No. 668,629 t 17 Claims. (Cl. 195-51) This invention relates to an improved method. for the biological dehydrogenation of steroids. It. relates'in particular to an improvement in the production of A steroids by the biological action of a fungus of the genus Septomyxa or by the action of the enzyme system associated with such fungi, in substrates comprising. novel combinations of steroids, at least one. of which: has an accelerating or otherwise unexpectedly beneficial. effect upon the bioconversion.

The substrates which have been found susceptible of bioconversion at rates substantially exceeding those customarily observed with Septomyxa may be characterized generally as polyhydroxy. steroids of the androstane or pregnane series containing the 3-keto-A configuration in the A ring.

Steroids which have demonstrated a capacity for achieving the unexpected increase in conversion rates when added. to the above indicated substrates in small quantities in. accordance with the present invention may be grouped as follows:

where R is hydroxy, carboxy or formyl, and mixtures of such compounds; and

where R is hydrogen or hydroxy. In many instances an increased conversion rate has been obtained through use of the A analogs of the above compounds. Among these highly effective assistants, 3-ketobisnor-4-cholen- 22-al-is to be preferred.

The process of preparing Il -steroids through the action ofthe fungus of the genus Septomyxa is described and claimed in copending application SN. 493,302, filed March 9, 1955, now abandoned. The present invention relates to the process of this prior application carried out in such a manner that the biological. action is accelerated or otherwise benefited.

According to this invention the process of l-dehydrogenation of steroids by the biological action of a fungus of the. genus Septomyxa is assisted or accelerated by the addition of a second steroid.v The presence of the bioconversion assistant as an additive to the substrate accelerates or assists the rate and/ or degree of conversion of a relatively slow converting steroid in an unexpected manner.

By. the method of the present invention, a variety of products can be produced which find use as valuable therapeutics per se as well as being useful as intermediates in the production of other therapeutic compounds. The n -steroids produced hereunder commonly have a lower melting point and enhanced physiological activity as compared with the corresponding l-saturated compounds. They find additional utility as intermediates in producing compounds in which it is desired to aromatize the A-ring.

Aspreviously stated, the process of preparing A steroids through the action of Septomyxa fungi is disclosed and claimed in a copending application. In carrying out that process, it has been frequently observed that the rate of bioconversion of some steroids is significantly slower than that noted in others. We have discovered that this slowness occurs. especially when the steroids exist in a state of relatively high purity in the substrate. Further detailed description of these slow steroids will be presented in the following description.

In contrast to the slow steroids, the majority of steroids encompassed by the invention described and claimed in the aforementioned copending application SN. 493,302 are relatively rapid, i.e., they are rapidly converted to the A -analog by the action of the Septomyxa organism. These relatively rapid steroids will likewise be described in more detail in the description which follows.

The dehydrogenation rate of the relatively slow steroids, in itself, is an economic disadvantage, inasmuch as large amounts of expensive equipment are involved and the time of highly skilled personnel is required to carry out the fermentation. The use of steroid assistants or accelerants is consequently of great economic significance in increasing the efficiency of a production unit. Their use, however, has the additional advantage that it tends to render the action of the Septomyxa organism selective in the l-dehydrogenation reaction. As disclosed in theaforementioned copending application, the l7-side chain of C or higher steroids can also be degraded by the action of Septomyxa to yield 17'-hy-droxy and 17- keto products, this being true especially when the substrate consists of a 20-oxygenated steroid. It is often desirable, however, that a C or higher steroid possessing a ZO-oXygenated side chain be l-dehydrogenated by Septomyxa without degrading the side chain. The use of assistants in our novelprocess significantly suppresses 17-side chain degradation while accelerating or assisting in l-dehydrogenation.

Typical illustrations of relatively slow steroids will be given in the paragraphs and examples which follow. The class of relatively slow steroids is inclusive of those mentioned as well as others, the term slow being defined as including those steroids which, present as the sole stera oidal constituent and in substantially pure form in the substrate, remain at least thirty percent unconverted in an aerobic fermentation by Septomyxa afinis after a period of 72 hours when added in a concentration of 0.25 gram per liter or more. Conversely, a relatively rapid steroid is one which remains less than thirty percent unconvented under the same conditions.

The categorization of slow steroids given herein will serve as a guide for most fermentations that are presently of special interest. To determine the character of a steroid not given in the list, it is convenient to carry out a pilot fermentation, e.g., by making up 100 milliliters of a medium containing one percent glucose, two percent corn steep liquor (sixty percent solids), and distilled or deionized water, sterilizing the medium for approximately one hour at fifteen pounds per square inch pressure and inoculating the medium thus prepared with a one-day growth of spores from Septomyxa afiinis, A.T.C.C. 6737. The sterilized and inoculated medium, preferably in a 250- milliliter Erlenmeyer flask, is then incubated at 24-28 degrees centigrade for a period of three days. The candidate steroid in substantially pure form is dissolved, for example, in dimethylformamide, propylene glycol or acetone and then added to the medium. Fermentation is allowed to proceed with moderate shaking for 72 hours at 24-28 degrees centigrade. At the end of the fermentation period, the mycelium and the culture liquor are extracted with methylene chloride, the extract evaporated .to dryness and the residue chromatographed. The ovhromatographic results are then compared with a control, and the degree of bioconversion determined. If more than thirty percent of the candidate steroid has remained unconverted, it is classifiable as a relatively slow steroid. If less than thirty percent remains unconverted, it is classifiable as a relatively rapid steroid. In the former case, conversion with Septomyxa afifm's can be accelerated by the addition to the substrate of a bioconversion assistant in accordance with this invention.

According to the aforementioned copending application S.N. 493,302 describing the fermentative l-dehydrogenation of steroids, the operational conditions and reaction procedure may be those already known in the art of steroid bioconversion, as illustrated by the Murray et al. U.S. Patent 2,602,769, utilizing, however, the action of a species of a fungus of the genus Septomyxa. The

operational conditions-and reaction procedure and details of the present invention are the same as those described in application S.N. 493,302, except that here the substrate is a relatively slow steroid to which has been added a bio conversion assistant.

Culture of the fungi for the purpose of practicing the present invention is in or on a medium favorable to the development of the fungi. The preferred media are those which permit quantity growth under aerobic conditions. Moist sol-id particulate media, such as bran, cereal grains, cereal grits, wood chips, shavings, sawdust, cornhusks, or fibrous material, such as copra, chestnuts, or lupine seeds can be used. These can be extracted with alcohol, ether or other organic solvents to remove objectionable contaminants and growth inhibitors prior to fermentation. The'carriers may optionally contain added growth fac tors and nutrients and can be used in layers or trays with or without auxiliary aeration, in towers as in the vinegar process, or under conditions of agitation as, for example, by tumbling in a rotating drum. Liquid media, such as brewers wort, are well adapted to use under an aerobiclayer or, more especially, aerobic submerged fermentation conditions. Preferably, the media should contain sources of available carbon, nitrogen and minerals, al-

though of course there can be significant growth and de-- velopment under less than optimum conditions.

Available carbon can be from carbohydrates, starches, gelatinized starches, dextrin, sugars, molasses as of cane, beet and sorghum, glucose, fructose, mannose, galactose, maltose, sucrose, lactose, pentoses, amino acids, peptones or proteins, the hexoses being preferred. Lower fatty acids, higher fatty acids, or fats are illustrative of other materials which provide assimilable carbon for the energy requirements of the fungi. Mixtures of various carbon sources are sometimes advantageous.

Nitrogen in assimilable form can be provided by soluble. or insoluble vegetable or animal proteins, soybean meal, lactalbumin, casein, egg albumin, peptones, polypeptides, amino acids, acid amides, urea, ammonium salts, ammonia trapped on base exchange resins or on zeolites, ammonium chloride, nitrates, sodium nitrate, potassium nitrate, or morpholine. Whey, distillers solubles, corn steep liquor, or yeast extract likewise have been useful.

As mineral constituents the media or menstruum can contain, naturally present or added, available aluminum, calcium, chromium, cobalt, copper, gallium, iron, magnesium, molybdenum, potassium, scandium, uranium, and vanadium. Sulfur can be provided by sulfates, alkyl sulfonates, sulfoxylates, sulfamates, sulfinates, free sulfur, hyposulfiate, persulfate, thiosulfate, methionine, cystine, cystein, thiamin or biotin. Phosphorous, preferably pentavalent, suitably in a concentration at or about 0.001 to 0.07 molar and particularly at or about 0.015 to 0.02 molar, can be present as ortho-, meta-, or pyrophosphate's, salts or esters, phytin, phytic acid, phytates, glycerophosphate, sodium nucleinate, and/or corn steep liquor, casein, lecithin or ovovitellin. Boron, iodine and selenium in traces can be advantageous. Boron, in the form of boric acid or sodium borate can be present or edded, especially after germination and early growth of thefungus.

Other accessory growth factors, vitamins, auxins and growth stimultants can be provided as needed or desired.

l benzoates, sulfites, penicilin, or tetracycline.

suspending or mycelial carriers such as filter earths, filter aids, finely divided cellulose, wood chips, bentonite, calcium carbonate, magnesium carbonate, charcoal, activated carbon or other suspendable solid matter, methyl cellulose, carboxymethyl cellulose, alginates or polyvinyl alcohol can be added to facilitate fermentation, aeration and filtration.

The selected species of fungus is grown on a medium suitably containing assimilable non-steroidal carbon, illustratively carbohydrates, such as dextrose; assimilable nitrogen, e.g., soluble or insoluble proteins, peptones or amino acids, and mineral constituents, such as sodium or ammonium phosphate and magnesium sulfate. The medium can desirably have a pH before inoculation of between about four to about seven, though a higher or lower pH may be used. A pH of between about five and about six is preferred for the growth of Septomyxa. Inocu1a tion of the fungi growth-supporting medium with the selected species of Septomyxa can be accomplished in any suitable manner. The Septomyxa grow over a temperature range from about 20 to about 38 degrees centigrade, with a temperature between about 20 to 35 degrees centigrade being preferred.

The development period of fungal growth required before the steroid to be converted is exposed to the fungus does not appear to be critical. For example, the steroid can be added either before thermal or other sterilization of the medium, at the time of inoculating the medium with a selected Septomyxa species, or at some other time, e.g., 24 to 96 hours later. The steroid to be fermented can be added at any suitable concentration, although for practical reasons steroid substrate at a concentration of up to about 0.5 gram per liter or even 0.8 gram per liter of medium is satisfactory and two grams per liter is operative, although higher concentra tion, depending on the particular steroid, can be used. The dition of steroid substrate to be converted can be accomplished in any suitable, manner to promote. a large. surface of contactof the steroid substrate with-thefungus,

such as by dispersing the steroidsubstrate, either alone or with a dispersing agent, or in solution in an organic solvent by mixing or homogenizing the steroid substrate with a fungal medium to form. a. suspension or dispersion of the steroid. Either submerged or surface culture procedures can be used with facility, although submerged culture is preferred. Alternatively, steroid converting enzymes of a growing fungus can be separated from the fungus or medium, then admixed with the steroid or a solution or dispersion thereof, and the mixture subjected to aerobic conditions to accomplish fermentation of the steroid. a

The temperature during the period of fermentation of the steroid may be that found suitable for fungal growth. It need be maintained only within such range as supports life, active growth, or the enzyme activity of the fungus.

While any form of aerobic incubation is satisfactory for the growth of the selectedfungus and fermentation ofilthe steroidsubstrate, the efficiency of steroid fermentation isrelated to aeration. and agitation. Therefore, aeration is usually controlled, as by agitation and/or blowing air through the fermentation medium. Aeration may be effected by surface culture or. under submerged fermentation conditions. Aerobic conditions include not only the use of air to introduce oxygen, but also other sources or mixtures containing oxygen in free or liberatable form. In using air as the aerating medium, a desirable rate of aeration is about four to twenty millimoles and particularly about nine to twelve millimoles of oxygen per hour per liter of beer as determined by the method of Cooper, Fernstrom and Miller, Ind. Eng. Chem. 36, 504 (1944). Aeration is suitably modified by using superatrnospheric or subatmospheric pressures, for example, thirty pounds per square inch or ten pounds per square inch absolute; Oxygen uptake can be facilitated by the presence ofvarious agents such as ascorbic acid, glutamic acid, citric acid, lactic acid, tyrosine, or tryptophane. The pH can be controlled by addition of alkali or phosphoric acid. The addition of excess calcium carbomate tomaintain a solid calcium carbonate residue has been found desirable. After completion of the steroid fermentation, the resulting fermented steroid is recovered from the fermentation reaction mixture. An especially advantageous manner of recovery involves extracting the fermentation reaction mixture, including the fermentation liquor and mycelia, with a water-immiscible organic solvent for steroids, e.g., methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichloroethylene, ether, amyl acetate, benzene, and the like. Alternatively, the fermentation liquor; and mycelium first can be separated and then separately extracted with suitable solvents. The mycelium canbe extracted with either water-miscible or water-immiscible solvents, acetone being effective. The fermentation liquor, freed of mycelia, can be extracted with water-immiscible solvents. The extracts can be combined, either before or after washing, with an alkaline solution, illustratively sodium bicarbonate, suitably dried, e.g., over anhydrous sodium sulfate, and the resulting fermented steroid. purified by recrystallization from organic solvents, by trituration or by chromatography in order to isolate the thus obtained steroid from the other fermentation products.

The cultural conditions of the present invention are thus substantially those described in copending application S.N. 493,302, with the exception that an additive is usedin the bioconversionstep. We have discovered that the process of the copending application frequently does not afford optimum yields of n -polyhydroxy steroids with Sepzomyxa ayfinis as the converting organism and that certain steroids added to the substrate act as bioconversion assistants, increasing the ultimate yield of desired product an unexpected and, as yet, undetermined manner.

Typical examples of relatively slow fermentation with Septomyxa a jtnis, particularly when. the starting materials exist in a state of relatively high purity in the substrate, are the production of 11,8,17a,2l-trihydroxyd,4-pregnadiene-3,20-dione (l-dehydrohydrocortisone) from 115, 1711,21 trihydroxy 4 pregnadiene 3,20-dione; 11B, l7a,2l trihydroxy 6 methyl 1,4-pregnadiene-3,20- dione (6-methyl l dehydrohydrocortisone) and the 6mand 6f3-epimers thereof from 11fi,17a,21-trihydroxy-6amethyl 4 pregnene-3,20-dio-ne;: 11fl,17a,21-trihydroxy- ZZ-methyl 1,4-pregnadiene 3,20-dione from 1lfl,17uc,2.l trihydroxy 2 methyl 4 pregnene-3,2 0-dione; 11oc,17oc, dihydroxy-],4 pregnadiene 3,20-dione from 1104,1711- dihydroxyprogesterone and 11B,l7u-dihydroxyprogesterone; 6l3,llu-dihydroxy l,4-pregnadiene-3,20-dione from 65,11a dihydroxyprogesterone; 11 3,17 5 dihydroxy-lA- pregnadiene 3,20-dione from 11fl,17fidihydroxyprogesterone; 1l/3,17ot-dihydroxy 17,8-methyl 9oz fluoro-1,4- pregnadien-3-one from 904 fiuoro hydroxy-lT- methylt'estosterone; 115,17a dihydroxy 17/3 methyl*9, 11B epoxy 1,4 androstadien 3 one from 9,11B-epoxy- 17-methyltestosterone and 1 1B,21-dihydroxy-1,4,17(20-)- pregnatrien 3 one from 11,8,21 dihydroxy-4,17(20)- pregnadien-3-one. Either the free hydroxy steroids or their esters may be used as starting steroid substrates. The resulting 1-dehydrogenation takes place in any event to produce the corresponding l-dehydro form of the starting material, recovered as the free alcohol.

Typical steroid compounds which increase the yield of the desired A analogs of compounds such as those mentioned above are progesterone, androstenedione, 3-ketobis nor-4-cholen-22-ol, 3-ketobisnor 4 cholen-22-al and 3- ketobisnor-4-cholen-22-oic acid.

The invention is carried out, as previously stated, in substantially the same manner as described in the prior copending application, adding the assistant material to the slow steroid either in the form of purified assistant, a mixture of the slow steroid and assistant, or a crude (unpurified) reaction product, either chemical or biological, containing the assistant as a principal steroid ingredient. The bioconversion assistant is conveniently added to the fermentation mixture along with the slow steroid that is to be converted by the Septomyxa organism, i.e., to the substrate steroid. The quantity of the assistant on a weight basis can vary over a wide range, a small but perceptible amount often being sufficient. For general purposes an effective weight ratio of assistant to substrate may be regarded as from 1:1 to 1:1000. In the majority of fermentations, the preferred weight ratio is from 1:1 to 1:100.

The assistants in our novel process frequently undergo bioconversion together with the substrate, such being the case, for example, where the assistant possesses a saturated linkage between carbon atoms 1 and 2 and/or where the assistant possesses a l7-acetyl or substitutedacetyl side chain. The assistant in bioconverted or unconverted form, as the case may be, is present in the final fermentation liquor. In those instances where separation of the converted substrate compound from the assistant compound is desirable, we prefer to employ those assistants Which are readily separable from the bioconverted substrate product by reason of their polar characteristics or other physical or chemical properties which render them separable by selective extraction, crystallization, chromatographic methods, and the like.

Instead of being added simultaneously with the substrate, the assistant can be added prior to that time, e.g., during the period of mycelial growth from spores. It can also be introduced subsequent to the addition of the substrate compound in those instances where it is desired to control the rate of conversion thereof, i.e., by accelerating the conversion at a later stage of the process.

As previously stated, the action of the assistant, i.e., its specific effect in the bioconversion systemcomprising Septomyxa aflinis, has not. ascyet been determined. As

Two grams of corn steep liquor containing about 48 percent solids and one gram of glucose were added to tap water to make 100 milliliters total liquid volume in a ZSO-milliliter Erlenmeyer flask. The pH was adjusted to 4.8 with sodium hydroxide solution and the resulting material sterilized for 45 minutes at fifteen pounds per square inch steam pressure. This medium was inoculated with Septomyxa afiinis and the inoculated flask then placed on a rotary shaker where incubation was allowed to proceed for two days with the flask being rotated around a one-half inch radius at approximately 330 rpm. A substrate of ten milligrams of hydrocortisone dissolved in propylene glycol was prepared and introduced slowly with constant swirling of the flask to minimize precipitation of the steroid. Ten milligrams of progresterone as the bioconversion assistant was dissolved in one milliliter of propylene glycol and added to the substrate-medium mixture; bioconversion was then permitted to proceed for four days, the flask being shaken throughout this period. At the end of this time, the steroid fraction was extracted with methylene chloride and the solvent removed by vacuum at room temperature. The extent of bioconversion was estimated by paper chromatographs on aliquots based on the equivalent of 200 micrograms of starting material. Hydrocortisone substrate was found to be entirely absent and l-dehydrohydrocortisone present to the extent of approximately 45 micrograms per 200 micrograms of starting material, or a conversion ratio (converted product to unconverted starting material) of 45 :0.

Substituting for the hydrocortiso-n substrate employed above other steroid substrates normmly slow in l-dehydrogenation with Septomyxa aflinis, namely, 6-methylhydrocortisone, 1l,8,17u,21-trihydroxy 2 methyl-4-pregnene- 3,20 dione, 11u,17a-dihydroxyprogesterone, 11,8,17a-dihydroxyprogesterone, 65,110 dihydroxyprogesterone, 1 113,175 dihydroxyprogesterone, 9a fluoro-l lfl-hyd'roxy- 17 methyltestosterone, 9,115 epoxy-l7 methyltestosterone, 115,21 dihydroxy-4,17(20)-pregnadien-3-one and the 21-esters of the above compounds containing a 21-hydroxy group, including hydrocortisone, is productive of l-dehydrogenated compounds at a rate significantly surpassing that observed in the absence of bioconversion assistants.

Portions of hydrocortisone substrate which had undergone fermentation under identical conditions and for the same period of time but without addition of a bioconversion assistant were examined chromatographically in the same manner as above to determine the extent of conversion. In three separate runs results were as follows:

Conversion ratio (micrograms of converted Results similar to those in the above table are obtained when any of the steroid substrates enumerated above are 8 subjected to the fermentative action of the Septomyxa fungus in the absence of a bioconversion assistant.

EXAMPLE 2 Conversion of hydrocortisone in the presence of 11a.-

hydroxyprogesterone The process of Example 1 was repeated except that five milligrams of lla-hydroxyprogesterone was employed as the assistant. Paper chromatographic analysis indicated a conversion ratio of 50:1.

Similar improvement in conversion ratios are obtained with the other steroid substrates of Example 1.

EXAMPLE 3 Conversion of hydrocortisone in the presence of 1 -dehydroprogesterone The process of Example 1 was repeated except that ten milligrams of 1-dehydroprogesterone was substituted for the assistant therein. On the basis of paper chromatographic estimations, a conversion ratio of 45:0 was ob: tained at the end of the bioconversion period.

Use of l-dehydroprogesterone in conjunction with the other steroid substrates described in Example 1 gives bioconversion ratios indicating similar improvement in the rate of l-dehydrogenation.

EXAMPLE 4 Conversion of hydrocortisone in the presence of 1 -dehydrotestosterone The process of Example 1 was repeated with the exception that ten milligrams of l-dehydrotestosterone was employed as the bioconversion assistant. A ratio of converted to unconverted product of 50:0 was noted, indicating a total absence of starting steroid at the conclusion of the fermentation period.

The addition of 1-dehydrotestosterone to the other steroid substrates enumerated in Example 1 is productive of similarly improved conversion ratios as compared with those obtained in the absence of the bioconversion assistants.

EXAMPLE 5 Conversion of hydrocortisone in the presence of 1,4-andr0stadiene-3,l 7-dione The process of Example 1 we repeated except that ten milligrams of 1,4-androstadiene-3,17-di0ne replaced the bioconversion assistant there employed. Results indicated a conversion ratio of 50:0, as in the preceding example.

The introduction of 1,4-androstadiene-3,17-dione into the other steroid substrates described in Example 1 yields bioconversion ratios indicating similar activity of this assistant in accelerating or facilitating l-dehydrogenation of these steroids.

EXAMPLE 6 Conversion of hydrocortisone in the presence of 3-ketobisnor-4-ch0len-22-ol EXAMPLE 7 Conversion of hydrocortisone in the presence of 3-ket0bisnor-1,4-ch0ladien-22-ol The process of Example 6- was repeated except that one milligram of 3-ketobisnor-1-,4choladien-22-ol, the A -analog of the assistant of Example 6, Was dissolved in one milliliter of propylene glycol and added to the hydrocortisone substrate as the assistant. At the conclusion of the three-day bioconversion period, no hydrocortisone substrate was identified, and 55 micrograms of l-dehydrohydrocortisone per 200 micrograms of starting material was estimated as the quantity of converted product, a conversion ratio of 55:0.

Substituting, for the substrate herein those described in Example 1 is productive of similarly improved bioconversion ratios as compared with those obtained in the absence of 3-ketobisnor-1,4-choladien-22-ol as the bioconversion assistant.

EXAMPLE 8 Conversion of hydrocortisone in the presence of 3-ketobisn0r-4-cholen-22-al The process of Example 6 was repeated except that one milligram of 3-ketobisnor-4-cholen-22-al was employed as the bioconversion assistant. Estimates from paper chromatographs indicated one microgram of unconverted hydrocortisone substrate per 200 micrograms of starting material remaining, with 55 micrograms of converted product per 200 micrograms of starting materialbeing identified for a conversion ratio of 55:1.

The use of 3-ketobisnor-4-cholen-22-al with the substrates listed in Example 1 gives similarly improved results as compared with bioconversion ratios obtained with substrates undergoing fermentation in the absence of the bioconversion assistant employed herein.

EXAMPLE 9 Conversion of hydrocortisone in the presence of 3-ket0bisnor-4-cholen-22-oic acid The process of Example 6 was repeated except that one. milligram. of 3-ketobisnor-4-cholen-22-oic acid was added. as the bioconversion assistant. Paper chromatograph estimates at the conclusion of the bioconversion period indicated two micrograms of hydrocortisone substrate per 200 micrograms of starting material and seventy micrograms of l-dehydrohydrocortisone per 200 micrograms of startingmaterial, a conversion ratio of 70:2.

When 3-ketobisnor-4-cholen-22-oic acid is added as the bioconversion: assistant to the steroid substrates indicated in Example 1 as being normally slow in dehydrogenation, results similar to those obtained with hydrocortisone as the substrate are noted.

EXAMPLE 10 Conversion of hydrocortisone in the presence of varying amounts of 3-ketobisn0r-4-ch0len-22-al' The procedure of Example 1 was repeated with the exception that before addition of the bioconversion assistant to-the= hydrocortisone substrate the substrate was divided into seven portions, each containing ten milligrams ofhydrocortisone. To six of these portions was added 3-l;etobisnor-4Pcholen-22-al (BNA) in varying amounts, the seventh remaining untreated. At the conclusion of the, bioconversion period the following results were observedi;

BNA added (milligrams) EXAMPLE 11 Conversion of 6-methylhydrocortisone in the presence of varying amounts of 3-ketobisnor-4-ch0len-22-al Conversion ratio (micrograms of converted to unconverted product per 200 micrograms of starting material) BNA added (milligrams) EXAMPLE 12 Conversion of 11,8,21-dihyclroxy-4,] 7(20) -pregnadien-3- one in the presence of varying amounts of 3-ket0bisnor- 4cholen-22-al The procedure of Example 1 was repeated except that four portions each containing ten milligrams of 115,21- dihydroxy 4,l7(20) pregnadien 3 one served as substrates. To each of three portions was added 3-keto bisnor-4-cholen-22-al (BNA) in varying amounts, and bioconversion was allowed to proceed. Results observed at the end of the bioconversion period were as follows:

Conversion ratio (micrograms of converted to unconverted product per 200 micrograms of starting material) BNA added (milligrams) Substitution of the bioconversion assistants described in Example 11 for the 3-ketobisnor"-4-cholen-22-al (BNA) of the present example likewise gives results indicative of accelerated conversion rates as compared with those obtained in the absence of bioconversion assistants.

EXAMPLE 13 1 Conversion of hydrocortisone in the presence of 3 ketobisnr-4-cholen-22-al Following the procedure of Example 1, a series of tests were conducted to determine the influence of the time of introduction of the additive with respect to the time of addition of the substrate to the growing culture. Hydrooortisone (HC in table below) was employed in 0.4 milliliter of dimethylformamide and the 3-ketobisnor-4-cholen-22-al (BNA) in 0.2 milliliter of acetone. Results are indicated as follows, conversions being estimated from paper chromatographs:

Time, HO, Time, Incuba- HG, A HG, BNA, Mg. BNA, Mg. HG, tion, Percent Percent Hours Hours Hours EXAMPLE 14 Conversion of hydrocortisone in the presence 0 4-androstene-3,17-tiione Again following the procedure of Example 1, a series of tests were conducted to determine, in the manner of Example 13, the relationship between the time of additions of the additive and substrate to the growing culture. Hydrocortisone was employed in 0.4 milliliter of dimethylformamide and the 4-androstene-3,17-dione in 0.2 milliliter of acetone. Results were as follows:

To a medium prepared as described in Example 1 and containing twenty milligrams of hydrocortisone substate per 100 milliliters of medium was added 0.1 milligram of progesterone per 100 milliliter of propylene glycol. Fermentation was allowed to proceed for three days. At the end of this bioconversion period the steroid fraction was extracted with methylene chloride and the extent of conversion estimated from paper chromatographs. Results indicated that 45 micrograms per 200 micrograms of starting material remained unconverted as hydrocortisone, forty micrograms per 200 micrograms of starting material being identified as l-dehydrohydrocortisone, a conversion ratio of 40:45,

The above procedure was repeated using progesterone in a concentration of 1.0 milligram per 100 milliliters of propylene glycol. The conversion ratio was increased EXAMPLE 16 Conversion of hydrocortisone in the presence of I-dehydrotestololactone The method of Example 15 was repeated using l-dehydrotestololactone as the bioconversion assistant in a concentration of 0.1 milligram per 100 milliliters of propylene glycol. The ratio of converted to unconverted material was estimated at 28:60.

12 Substituting for the above quantity of l-dehydrot'es tolo lactone a concentration of one milligram per milli liters of propylene glycol was productive of a converted to unconverted product ratio of 45:40.

When the steroid substrates described in Example 1 are substituted for the hydrocortisone substrate of the present example, similar bioconversion ratios are obtained.

EXAMPLE 17 Conversion of hydrocortisone in the presence of 3-ketm bisnor-4-cholen-22-al The method of Example 15 was repeated with 0.1 milligram of 3-ketobisnor-4-cholen-22-al per 100 milliliters of propylene glycol. A converted to unconverted product ratio of 55:2 was obtained. e

Increasing the concentration of the assistant to one milligram per 100 milliliters of propylene glycol gave only trace amounts of unconverted productas compared with forty micrograms of l-dehydrohydrocortisoneper 200 micrograms of starting material.

EXAMPLE 18 Conversion of hydrocortisone in the presence of 3-ketobisnor-4-ch0len-22-0ic acid The method of Example 15 was repeated with 0.1 milligram of 3-ketobisnor-4-cholen-22-oic acid as the assistant. There was produced a converted product bearing a ratio to unconverted material of 50:35.

Substituting one milligram of this assistant per 100 milliliters of propylene glycol for the smaller amount was productive of a converted to unconverted product ratio of 45:2.

EXAMPLE 19 Conversion of hydrocortisone in the presence of 3-ketobisnor-I,4-choladien-22-oic acid EXAMPLE 20 Conversion of hydrocortisone in the presence of 3-ketdbisnor-I ,4 -choladi en-22 ol The method of Example 15 was repeated with 3-ketobisnor-l,4-choladien-22-ol, 0.1 milligram per 100 milliliters of propylene glycol, as the bioconversion assistant. The ratio of converted to unconverted product was 45 :2.

Substituting one milligram of the above assistant per 100 milliliters of propylene glycol was productive of complete conversion, no trace of the starting hydrocortisone being observed, as compared with thirty micrograms per 200 micrograms of starting material being identified as l-dehydrohydrocortisone, for a conversion ratio of 30:0.

EXAMPLE 21 Conversion of 6oi-fluorohya'rocortisone in the presence of 3-ketobisnor-4-ch0len-22-al septomyxa affinis was grown for 72hours in a shaken culture of corn steep glucose medium. Ten liters of a sterile medium containing lard oil as an antifoaming agent was inoculated with 500 milliliters of this growth.

The pH of the inoculated medium was 4.6, and at the nd of 24 hours, with continuous agitation and aeration, the pH had risen to 5.2. At this point 2.5 grams of oa-fluoro 115,170; dihydroxy-Z1-acetoxy-4-pregnene- 3,20-dione dissolved in fifteen milliliters of dimethylformamide containing 0.5 gram of 3-ketobisnor-4-cholen- 22-al as the bioconversion assistant was added to the growth medium. Additional lard oil was added at 48 hours to control foaming. After 72 hours the pH of the culture was 7.5. The mycelium was separated by filtration and the aqueous phase extracted four times with three liters each of a mixture containing approximately one liter of methylene chloride and 500 milliliters of ethyl acetate. An aliquot sample of the solvent extract was examined by paper chromatography and found to contain no 21-acetate starting material and only a slight trace of hydrolyzed but unconverted 21-alcohol. Extraction yielded a residue weighing 2.939 grams which was purified by chromatography over Florisil using ethylene chloride with increasing amounts of acetone. Recrystallization from methylene chloride gave 548 milligrams of material melting at 202 to 204 degrees centigrade and having [ab plus 92 degrees. Ultraviolet and infrared absorptions confirmed the structure, and analysis was as follows:

Calculated for C H O C, 66.65; H, 7.10. Found: C, 66.68; H, 7.19.

As a control study in the above experiment, dehydrogenation of the same starting material was attempted without the use of 3-ket0bisnor-4-cholen-22-al. No identifiable dehydrogenated product was obtained, and only the free alcohol was found to be present.

In conjunction with the foregoing examples it should be particularly noted that hydrocortisone, 6-methylhydrocortisone, and numerous other steroids present a difficult problem at best insofar as dehydrogenation at the 1-position by conventional methods is concerned. Experience has indicated, as pointed out previously, that these compounds are either incompletely or slowly de hydrogenated by methods known in the art, including the procedure disclosed in the copending application Serial No. 493,302.

The mechanism behind the phenomenon constituting the present invention is not readily understood. The lack of consistency in the degree of effectiveness of closely related compounds in facilitating the dehydrogenation is apparent from a review of the examples cited herein. Thus it cannot be said that a compound effective as an assistant in promoting the bioconversion must itself necessarily be converted in the process, for, as indicated in the foregoing examples, a number of compounds already dehydrogenated at the 1-position have been found highly effective in this regard. The present invention therefore embraces in its broadest aspect the addition to a steroid substrate undergoing biological dehydrogenation at the 1-position of a second steroid or mixture in which the said second steroid predominates for the purpose of accelerating, stimulating or in some manner assisting the bioconversion, whereby the desired product is obtained in greater yield, an established yield is realized in a significantly shorter fermentation period, or both. The addition of these bioconversion assistants, as they have been termed herein, has been shown to be admirably suited to facilitation of l-dehydrogenation of 3-keto-A -polyhydroxy steroids of the pregnane and androstane series.

It is to be understood that the present invention is not to be construed as limited to the exact details of operation or exact compounds or reactions hereinbefore shown and described, as obvious modifications and equivalents will be apparent to those skilled in the art; the invention is therefore to be limited only by the scope of the appended claims.

We claim:

1. In a process which comprises growing a fungus of the genus Septomyxa under aerobic conditions in the presence of a nutrient medium and a steroid to produce 14 the resulting A steroid, the improvement whichcomprises: contacting the steroid substrate during fermentation with a second steroid, whereby to accelerate l-dehydrogenation of the substrate steroid.

2. The improvement in the process of 1-dehydrogenation of a normally slow steroid by the action of Sept0= myxa afiinis which comprises: contacting the steroid substrate during fermentation with a small but perceptible amount of a second steroid, whereby to accelerate l-dehydrogenation of the substrate steroid, and thereafter separating the resulting A steroid.

3. The improvement in the process of l-dehydrogenat'ion of a normally slow steroid selected from the group consisting of steroids of the androstane and pregnane series by the action of Septomyxa afiinis which comprises: contacting the steroid substrate during fermentation with a small but perceptible amount of a second steroid, whereby to accelerate l-dchydrogenation of the substrate steroid, and thereafter separating the resulting A steroid.

4. The improvement in the process of l-dehydrogenation of a normally slow steroid selected from the group consisting of steroids of the androstane and pregnane series by the action of Septomyxa afifnis which comprises: contacting the steroid substrate with a bioconversion assistant selected from the group consisting of (1) compounds of the following formula:

wherein R is a member selected from the group consisting of keto and hydroxy, (2) compounds selected from the group consisting of steroids of the following formula:

CH R

CH 0H3 H 0 wherein R is a member selected from the group consisting of hydroxy, carboxy and formyl, (3) compounds selected from the group consisting of progesterone and ll-hydroxyprogesterone, and (4) mixtures thereof.

5. The improvement in the process of l-dehydrogenation of a normally slow 3-keto-A -steroid by the action of Septomyxa ajfinis which comprises: contacting the steroid substrate during fermentation with a small but perceptible amount of a streroid of the following formula:

R CH wherein R is a member selected from the group consisting of keto and hydroxy, whereby to accelerate l-dehydrogenation of the substrate steroid, and thereafter separating the resulting A steriod.

9,. The improvement of claim 8 in which the steroid,

substrate is hydrocortisone.

10. The improvement in the process of l-dehydrogenation of a normally slow 3-keto-A -steroid by the action of Septomyxa afiinis which comprises: contacting the steroid substrate during fermentation with a small but perceptible amount of a steroid of the following formula:

wherein R is a member selected from the group consisting of hydroxy, carboxy, and formyl, whereby to accelerate l-dehydrogenation of the substrate steroid, and thereafter separating the resulting A steroid.

11. The improvement of claim in which the bioconversion assistant is 3-ketobisnor-4-cholen-22-a1.

12. The improvement of claim 11 in which the steroid substrate is 6-rnethylhydroc0rtisone.

13. The improvement of claim 10 in which the bioconversion assistant is 3-ketobisnor-4-cholen-22-oic acid.

14. The improvement in the process of l-dehydrogenation of a normally slow 3-keto-A -steroid by the action of Septomyxa afiinis which comprises: contacting the steroid substrate during fermentation With a small but perceptible amount of progesterone, whereby to accelerate l-dehydrogenation of the substrate steroid, and thereafter separating the resulting A -steroid.

15. The improvement of claim 14 in which the steroid substrate is hydrocortisone.

16. The improvement in the process of l-dehydrogenw tion of a normally slow steroid by the action of Septomyxa afiinis which comprises: contacting the steroid sub-- strate during fermentation with a small but perceptible amount of a second steroid, whereby to accelerate I-dehydrogenation of the substrate steroid. r

17. The improvement in the process for the l-dehydro genation of 6a-methylhydrocortisone to produce methyl-A -hydrocortisone by the action of Septomyxa afiinis which comprises: contacting the 6u-methylhydrocortisone substrate during fermentation with a small but perceptible amount of 3-ketobisnor-4-cholen-22-al.

References Cited in the file of this patent Kahnt et al.: Experientia, 8, 1952, pp. 171, 172.

Finch et al.: Mfgr. Chemist, XXV, 6, June 1954, pp. 248, 249.

Thorn: Ann. New York Academy of Science, 60, 1, October 54, pp. 5, 24 and 26.

A hi I; 

1. IN A PROCESS WHICH COMPRISES GROWING A FUNGUS OF THE GENUS SEPTOMYXA UNDER AEROBIC CONDITIONS IN THE PRESENCE OF A NUTRIENT MEDIUM AND A STEROID TO PRODUCE THE RESULTING $1 STEROID, THE IMPROVEMENT WHICH COMPRISES: CONTACTING THE STEROID SUBSTRATE DURING FERMENTATION WITH A SECOND STEROID, WHEREBY TO ACCELARATE 1-DEHYDROGENATION OF THE SUBSTRATE STEROID. 